Two small, cysteine-rich and cationic antifungal proteins from Penicillium chrysogenum: A comparative study of PAF and PAFB.

The filamentous fungus Penicillium chrysogenum Q176 secretes the antimicrobial proteins (AMPs) PAF and PAFB, which share a compact disulfide-bond mediated, ß-fold structure rendering them highly stable. These two AMPs effectively inhibit the growth of human pathogenic fungi in micromolar concentrations and exhibit antiviral potential without causing cytotoxic effects on mammalian cells in vitro and in vivo. The antifungal mechanism of action of both AMPs is closely linked to - but not solely dependent on - the lipid composition of the fungal cell membrane and requires a strictly regulated protein uptake into the cell, indicating that PAF and PAFB are not canonical membrane active proteins. Variations in their antifungal spectrum and their killing dynamics point towards a divergent mode of action related to their physicochemical properties and surface charge distribution. In this review, we relate characteristic features of PAF and PAFB to the current knowledge about other AMPs of different sources. In addition, we present original data that have never been published before to substantiate our assumptions and provide evidences that help to explain and understand better the mechanistic function of PAF and PAFB. Finally, we underline the promising potential of PAF and PAFB as future antifungal therapeutics.

Splice variants reveal the region involved in oxygen sensing by recombinant human L-type Ca(2+) channels.

Regulation of vascular smooth muscle Ca(2+) channels by oxygen tension contributes importantly to hypoxic vasodilatation. We previously described the inhibitory effects of hypoxia on the recombinant human cardiac L-type Ca(2+) channel alpha(1C) subunit (hHT isoform) expressed in HEK 293 cells. We now demonstrate that hypoxia inhibits only one of the three naturally occurring splice variants of this channel that differ only in the C-terminal domain, permitting identification of a 71-amino acid insert in the C-terminal region of the channel that confers oxygen sensitivity. Selective restriction of the spliced insert allowed determination of a 39-amino acid region essential for oxygen sensing. This represents the first identification of the structural region of an ion channel required for sensing changes in oxygen tension.

[Radial optic neurotomy for central retinal vein occlusion -- how deep should it be?]. / Wie tief muss die radiäre Optikusneurotomie zur Therapie des retinalen Zentralvenenverschlusses sein?

PURPOSE: Most of the studies on radial optic neurotomy (RON) have not defined the depth of the incision. Complications following a deeper incision have been described. This histological study was performed to evaluate the required depth for RON. METHODS: Serial sections of the area of the optic nerve head were performed in 19 eye bank eyes. The distance between the inner surface of the optic disc and the outer limit of the cribriform plate was measured. Ten additional eye bank eyes underwent 2 mm deep experimental RON using the Spaide CRVO Knife (DORC, Netherlands). The cutting depth was assessed histologically by serial cuts. RESULTS: The distance between the inner surface of the disc and the outer limit of the cribriform plate measured 1.35+/-0.3 mm (shrinkage-revised value: 1.45 mm). The experimental RON showed cutting depths of 1.53+/-0.3 mm (shrinkage-revised value: 1.65 mm). CONCLUSION: Based on normal eyes, a cutting depth of 1.45 mm is sufficient to cut through the cribriform plate. This might change during central retinal vein occlusion because possible papillary edema due to central retinal vein occlusion has to be considered. Even under controlled experimental conditions RON leads to great variation in incision depths. The development of a knife with a fixed penetration depth would be helpful.

Effect of succinate on aminopyrine N-demethylase activity.

Succinate stimulated the aminopyrine N-demethylase activity in the presence of rate-limiting concentrations of NADPH in the crude mitochondrial preparation, as well as in a reconstituted system containing microsomes and microsome-free mitochondria. The increase in enzyme activity was accompanied by a decrease of the apparent Km of NADPH. The stimulating effect of succinate was counteracted by malonate, rotenone and pentachlorophenol. No significant formaldehyde oxidation was exhibited by the crude mitochondrial preparation under the conditions of N-demethylase assay. Enhancement of the N-demethylase activity by succinate is supposed to be due to a mitochondrial-microsomal interaction which may play a role in the regulation of drug metabolism.

A Ca(2+)-dependent transgenic model of cardiac hypertrophy: A role for protein kinase Calpha.

BACKGROUND: Calcium imbalances have been implicated as an underlying mechanism of human cardiac dysfunction. The voltage-dependent calcium channel plays a critical role in calcium regulation in the heart. Thus, aberrant calcium signaling arising from this channel could initiate the calcium imbalances observed in heart failure. In the present study, we used a transgenic mouse with an increased number of L-type calcium channels to identify the role of an increased, sustained ingress of calcium as an initiator of hypertrophy. METHODS AND RESULTS: Whole-heart histology and electrophysiology in isolated cardiomyocytes identified calcium-channel overexpression in the hearts of transgenic mice. Calcium-channel density was increased in 2-, 4-, and 8-month-old transgenic cardiomyocytes. Ventricular fibrosis, damage, and remodeling became more pronounced as the transgenic mice aged. Apoptosis was also present in transgenic hearts at 8 months of age. Increased protein kinase Calpha activation was elevated before the development of hypertrophy and failure. CONCLUSIONS: Transgenic mice developed hypertrophy and severe cardiomyopathy as a function of age, thus confirming that changes in channel density are sufficient to induce disease. The small, sustained increase in the ingress of Ca(2+) through the calcium channel elevated protein kinase Calpha before the development of hypertrophy, suggesting that protein kinase Calpha plays an important role in triggering hypertrophy.

Use of transgenic mice to study voltage-dependent Ca2+ channels.

During the past decade a great number of genes encoding high- and low-voltage-dependent Ca(2+) channels and their accessory subunits have been cloned. Studies of Ca(2+) channel structure-function relationships and channel regulation using cDNA expression in heterologous expression systems have revealed intricate details of subunit interaction, regulation of channels by protein kinase A (PKA) and protein kinase C (PKC), drug binding sites, mechanisms of drug action, the ion conduction pathway and other aspects of channel function. In recent years, however, we have arrived at the brink of an entirely new strategy to study Ca(2+) channels by overexpressing or knocking out genes encoding these channels in transgenic mice. In this article, various models of gene knockout or gene overexpression will be discussed. This new approach will reveal many secrets regarding Ca(2+) channel regulation and the control of Ca(2+)-dependent cellular processes.

Molecular determinants of Ca2+ channel function and drug action.

Molecular cloning has revealed the existence of six high-voltage activated Ca2+ channel types. Expression studies have shown that basic high-voltage activated channel function, which is typical for the L-(skeletal muscle, cardiac muscle and neuroendocrine tissue), N-, P-, Q- and R-type channels is carried by the corresponding alpha 1 subunits. Auxiliary subunits, such as alpha 2/delta and beta, modulate the kinetics of activation, inactivation, current density and drug binding, thereby creating considerable potential for multiple Ca2+ channel functions. Glutamic acid residues in the pore (P) loops are molecular components that impart high selectivity for Ca+. Binding or pharmacologically active sites for Ca2+ channel drugs have been localized on various segments of the alpha 1 subunit in close proximity to the pore lining. In this article, Gyula Varadi and colleagues review the roles of the different subunits in Ca2+ channel function and suggest that Ca2+ channel drugs act by blocking or, in some cases, activating channel function via binding directly or indirectly to the pore structure of the channel.

Non-supralethal mitobronitol/cytarabine/cyclophosphamide conditioning without irradiation before bone marrow transplantation for accelerated chronic granulocytic leukemia: apparent absence of acute graft-versus-host disease.

Cytostatic chemotherapy instead of supralethal total body irradiation (TBI) has been increasingly used as an alternative myeloablative regimen before bone marrow transplantation (BMT). While irreversible azoospermia/amenorrhoea seems to occur less frequently with such conditioning, graft-versus-host disease (GVHD) remains unaffected. Five-year disease-free survival in accelerated chronic granulocytic leukemia (CGL), after BMT with matched sibling grafts has been 0.10-0.30. Mitobronitol, cytosine arabinoside, and cyclophosphamide were used for conditioning. Patients were transplanted with unmanipulated HLA/MLC identical sibling bone marrow. For recovery, a pathogen-low room was available without air filtering and laminar airflow. Seven of eight accelerated-CGL patients were engrafted: full allogeneic reconstitution was detected in four and mixed chimerism in three patients. Five out of the seven engrafted patients survived at least nine months (median = 42 months), two are considered cured (8-9 years survival). The four leukemia-free survivors displayed full allogeneic reconstitution and presented symptoms of chronic GVHD. One patient became a genetically verified father. Acute GVHD and veno-occlusive liver disease (VOLD) were absent in all patients, diffuse interstitial pneumonitis (IP) occurred in one case. Non-supralethal conditioning with mitobronitol/cytarabine/cyclophosphamide in accelerated-CGL allows allogeneic bone marrow reconstitution with survival and cure rates comparable to those achieved with other protocols using TBI or busulphan conditioning. Unlike the latter treatments, however, our protocol leads to fewer transplant-related complications including acute GVHD, IP, VOLD, and azoospermia/amenorrhoea.

T cell recognition of the posttranslationally cleaved intersubunit region of influenza virus hemagglutinin.

The influenza virus hemagglutinin is synthesized as a single polypeptide chain, but upon maturation it will posttranslationally be modified by a host cell related trypsin-like enzyme. The enzymatic cleavage attacks the so-called intersubunit region of the molecule giving rise to covalently linked HA1 and HA2 subunits. An I-Ed-restricted T cell epitope was identified in the highly conserved intact intersubunit region of the influenza virus hemagglutinin. T cell recognition of a 25-mer synthetic peptide comprising the intact intersubunit region does not require further processing and the elimination of the intervening Arg residue coupling the fusion peptide to the C-terminal segment of HA1 does not abolish the T cell activating capacity. The fine specificity pattern of a T cell hybridoma similar to that of the polyclonal T cell response demonstrates that a single T cell receptor is able to recognize peptides of different sizes representing not only the uncleaved but also the cleaved form of this hemagglutinin region. Based on specificity studies the epitope was localized to the C-terminal 11 amino acids of the HA1 subunit. The cross-reactivity of peptide-primed T cells with influenza virus infected antigen-presenting cells shows that fragments comprising the identified epitope of the intersubunit region can be generated as a result of natural processing of the hemagglutinin molecule. As antigen-presenting cells are lacking the enzyme which is responsible for the posttranslational modification of newly synthesized hemagglutinin molecules, the role of immature viral proteins in immune recognition is discussed.

Graft vs autoimmunity following allogeneic non-myeloablative blood stem cell transplantation in a patient with chronic myelogenous leukemia and severe systemic psoriasis and psoriatic polyarthritis.

OBJECTIVE: No specific therapy exists for autoimmune diseases caused by self-reactive lymphocytes. As shown in experimental animals, which led to pilot clinical studies, elimination of self-reactive lymphocytes can be accomplished with high-dose chemoradiotherapy, followed by autologous stem cell transplantation, by re-establishment of unresponsiveness to self antigens of newly generated lymphocytes, due to a mechanism of central clonal deletion. We hypothesized that self-reactive lymphocytes causing autoimmune disease may be successfully eliminated by highly immunosuppressive yet not necessarily myeloablative conditioning in conjunction with allogeneic blood stem cell transplantation, since immunocompetent alloreactive lymphocytes of donor origin can effectively eliminate residual host-type hematopoietic cells, self-reactive lymphocytes included, by a mechanism that resembles graft-vs-leukemia (GVL) effects. The present report is an attempt to confirm the existence of graft-vs-autoimmunity (GVA) effects in parallel with amplification of the alloreactive potential of donor lymphocytes following allogeneic non-myeloablative stem cell transplantation (NST). METHODS: We identified a patient with severe psoriatic arthritis who also had Philadelphia (bcr/abl) positive chronic myelogenous leukemia and therefore was fully eligible for NST. Both diseases responded initially to non-myeloablative conditioning involving fludarabine 30 mg/m2 x 6, anti-T-lymphocyte globulin 10 mg/kg X 4, and busulfan 4 mg/kg x 2. RESULTS: The initial NST procedure was uneventful and resulted in elimination of all signs of autoimmunity (psoriasis and arthritis). Recurrence of polyarthritis and exacerbation of psoriasis were observed in parallel with a significant increase in the proportion of male (host) DNA, and 5% of the mitoses were bcr/abl positive, indicating an increase in the clone of CML. Both bcr/abl-positive cells identified by RT-PCR and psoriatic arthritis were successfully eliminated following discontinuation of anti-GVHD prophylaxis with cyclosporine A (CSA), which resulted in activation of the alloreactive potential of donor T cells, accompanied by graft-vs-host disease (GVHD), suggesting the existence of GVA effects. RT-PCR for bcr/abl remains consistently negative for nearly 3 years, and all DNA remains donor type. CONCLUSIONS: The response of autoimmune disease manifestations to GVA effects in parallel with elimination of all host-derived hematopoietic cells supports our working hypothesis that autoimmune diseases caused by self-reactive lymphocytes may be effectively treated by elimination of alloreactive self-reactive lymphocytes following induction of host-vs-graft tolerance, in analogy with replacement of malignant or genetically abnormal host cells following DLI. It is therefore suggested that intentional GVA effects may be inducible by DLI following a conventional or preferably safer non-myeloablative regimen in recipients with life-threatening autoimmune diseases resistant to conventional modalities. Adoptive immunotherapy of autoimmunity may thus involve a two-step procedure: first, inducing host-vs-graft and graft-vs-host transplantation tolerance through a transient stage of mixed chimerism; second, inducing controlled GVA effects, initially by discontinuation of CSA and then, if indicated, by late outpatient DLI to eradicate residual hematopoietic cells of host origin.

Second allogeneic stem cell transplantation using nonmyeloablative conditioning for patients who relapsed or developed secondary malignancies following autologous transplantation.

OBJECTIVE: Second allogeneic stem cell transplants for hematological malignancies are associated with a high incidence of transplant-related mortality due to the cumulative incidence of toxicity of the high-dose chemoradiotherapy traditionally used as an essential component of the conditioning. We have demonstrated previously that nonmyeloablative conditioning for primary allogeneic transplants from both sibling and unrelated donors results in minimal transplant-related toxicity and excellent stem cell engraftment. This study explores the possibility of using nonmyeloablative conditioning to minimize transplant-related toxicity in patients who have undergone second allogeneic transplants. PATIENTS AND METHODS: Twelve high-risk, heavily treated patients-five with acute myelogenous leukemia (AML); five with non-Hodgkin's lymphoma (NHL); one with Burkitt's lymphoma, and one with acute lymphoblastic leukemia (ALL)-underwent second allogeneic nonmyeloablative stem cell transplantation (NST) from human leukocyte antigen (HLA)-matched donors, 29 (median) (range 3-57) months following their first transplantation procedure. The conditioning consisted of fludarabine 30 mg/m(2) daily for 6 days, busulfan 4 mg/kg daily for 2 days, and anti-T-lymphocyte globulin 10 mg/kg daily for 4 days. Anti-graft-vs-host disease (anti-GVHD) prophylaxis consisted of cyclosporine A alone, 3 mg/kg. RESULTS: Engraftment was observed in all recipients, with complete and stable chimerism. None of the patients developed veno-occlusive disease of the liver or multi-organ failure. Five very high-risk patients with NHL (n = 3), Burkitt's lymphoma (n = 1), and AML (n = 1) relapsed 2 to 6 months post-transplant, and four of them died. Six patients appear to be disease-free after median follow-up of 23 months. One additional patient died from grade IV hemorrhagic cystitis. Actuarial survival and disease-free survival at 34 months are 56% and 50% respectively, with 95% confidence interval (25-78%). CONCLUSION: These results suggest that nonmyeloablative conditioning significantly reduces transplant-related toxicity, thus making a second transplant feasible.

Low-intensity conditioning is sufficient to ensure engraftment in matched unrelated bone marrow transplantation.

OBJECTIVE: Matched unrelated bone marrow transplantation (BMT) for patients with hematological malignancies is associated with a high incidence of transplant-related complications due to high doses of chemoradiotherapy administered pre-BMT to ensure engraftment. The aim of this study was to investigate the feasibility of low-intensity conditioning for BMT from matched unrelated donors. MATERIALS AND METHODS: Sixteen patients with hematologic malignancies underwent non-T-cell-depleted BMT following a low-intensity conditioning regimen consisting of fludarabine monophosphate 30 mg/m(2)/day for 6 days, busulfan 4 mg/kg/day for 2 days, anti-T lymphocyte globulin 10 mg/kg/day for 4 days. Seven of the patients suffered from chronic myelogenous leukemia, four from acute lymphoblastic leukemia, four from acute myelogenous leukemia, and one from Ki-1 non-Hodgkin's lymphoma. Three of the patients had secondary leukemia and two were post-autologous BMT (ABMT). All patients were transplanted from fully matched unrelated donors. RESULTS: Fifteen of the 16 patients had 100% donor chimerism; no graft rejection was observed. None of the patients developed >Grade II veno-occlusive disease, sepsis, multiorgan failure, or renal or pulmonary toxicity. Four patients died posttransplant; one of thrombocytopenia and severe hemorrhagic cystitis, one of central nervous system toxicity, one of Grade IV graft-vs-host disease, and one following relapse (9 months post-BMT). Survival and disease-free survival at 36 months are 75% (95% confidence interval 46-90%) and 60% (95% confidence interval 30-80%), respectively. CONCLUSION: These results indicate that low-intensity conditioning is sufficient to ensure stable engraftment of bone marrow grafts in a matched unrelated setting.

Engraftment of marrow allografts treated with Campath-1 monoclonal antibodies.

We have analyzed the factors associated with engraftment in 216 recipients of T-cell depleted allogeneic HLA identical sibling marrow transplants using Campath 1 monoclonal antihuman lymphocyte (CD52) antibodies. The patient population consisted of 168 patients with hematologic malignancies, 26 with severe aplastic anemia (SAA), and 22 with hemoglobinopathies, half of whom received marrow treated in vitro with Campath-1M (IgM) and half received marrow with Campath-1G (IgG2b isotype). Patients with durable engraftment had fast hematopoietic recovery: SAA patients reached ANC > 0.5 x 10(6)/L on Day 14; those with leukemia attained ANC > 0.5 x 10(6)/L on Days 18, 17, and 15 for ANLL, ALL and CML respectively, while patients with thalasemia reached ANC > 0.5 x 10(6)/L on Day 21. Overall, 24 patients (17 with leukemia, 4 with SAA, and 3 with thalassemia) suffered graft failure: 10 patients (all grafted with Campath-1M) rejected their grafts, while 14 others (9 grafted with Campath-1M, and 5 with 1G isotype) never engrafted (p = 0.009). Multivariate analysis revealed that neither pretransplant protocol, nor stage of disease or type of antibody used, donor sex and ABO match had any impact on engraftment. The variables favorably associated with engraftment were older age (p = 0.030, RR = 1.016) and CFU-GM number (p = 0.013, RR = 1.001). Patients with ANLL or SAA had a better chance to engraft (p = 0.027, RR = 1.400; and p = 0.003, RR = 2.677, respectively) compared to patients with thalassemia (p = 0.001, RR = 0.551). A higher concentration of Campath-1 antibody in vitro and in vivo adversely affected engraftment. Our data show that satisfactory engraftment can be achieved in patients transplanted with Campath-1 treated marrow allografts. However, despite the measures undertaken to prevent rejection, graft failure still poses a problem. Further pretransplant immunosuppression and perhaps more selective T-cell depletion may reduce the increased graft failure in these patients.

Developmental regulation of expression of the alpha 1 and alpha 2 subunits mRNAs of the voltage-dependent calcium channel in a differentiating myogenic cell line.

The voltage-dependent calcium channel (VDCC) in skeletal muscle probably plays a key role in transducing membrane charge movement to the calcium release channel. We report here that the expression of VDCC alpha 1 and alpha 2 mRNAs is developmentally regulated in differentiating C2C12 myogenic cells. The alpha 1 mRNA is not detectable in the myoblast form of C2C12 cells while its expression is induced 20-fold in differentiated myotubes. In contrast, the alpha 2 mRNA is weakly expressed in myoblasts but is also induced upon myogenic differentiation.

Characterization of beta subunit modulation of a rabbit cardiac L-type Ca2+ channel alpha 1 subunit as expressed in mouse L cells.

Functional properties of a rabbit cardiac alpha 1 Ca2+ channel subunit (CARD alpha 1) were investigated using the patch-clamp technique in mouse L cells, a recipient cell line which is devoid of any Ca2+ channel subunits. Cell lines resulting from stable transfection of the CARD alpha 1 subunit as well as in coexpression with a beta subunit (CARD alpha 1 beta) derived from skeletal muscle (SKM beta) were characterized. The results show that while the CARD alpha 1-Ca2+ channel activity is negligible, the Ba2+ current density is dramatically increased in the presence of beta subunit (approximately 20-fold). CARD alpha 1- and CARD alpha 1 beta-Ba2+ currents were both sensitive to the 1,4-dihydropyridine (DHP) agonist, Bay K 8644 (5- to 8-fold increase). Activation kinetics of CARD alpha 1- and CARD alpha 1 beta-Ba2+ currents were comparable. The inactivation time-course was faster (3- to 4-fold) for CARD alpha 1 beta-Ba2+ currents. We conclude that the main role of the beta subunit in heart is to modulate the L-type current density and present several lines of evidence that SKM alpha 1 and CARD alpha 1 are differentially regulated by the beta subunit.

Evidence for the existence of a cardiac specific isoform of the alpha 1 subunit of the voltage dependent calcium channel.

Biochemical, pharmacological and electrophysiological evidence implies the existence of tissue specific isoforms of the L-type VDCC. The alpha 1 and alpha 2 subunits of the skeletal muscle calcium channel have been previously cloned and their amino acid sequence deduced. Here we report the isolation and sequencing of a partial cDNA that encodes a heart specific isoform of the alpha 1 subunit. The amino acid sequence deduced from this part cDNA clone shows 64.7% similarity with the skeletal muscle alpha 1 subunit. Northern analysis reveals 2 hybridizing bands, 8.5 and 13 kb, in contrast to one 6.5 kb band in the skeletal muscle. Selective inhibition of mRNA expression in Xenopus oocytes by complementary oligodeoxy-nucleotides derived from the heart clone provides further evidence that the cDNA corresponds to an essential component of the VDCC. These data further support the existence of tissue-specific isoforms of the L-type VDCC.

Endogenous cardiac Ca2+ channels do not overcome the E-C coupling defect in immortalized dysgenic muscle cells: evidence for a missing link.

The expression of subunit genes of the Ca2+ channel complex was studied in differentiating, immortalized mouse mdg cells. These cells expressed alpha 1 and alpha 2/delta transcripts of the skeletal muscle Ca2+ channel genes, a cardiac Ca2+ channel alpha 1 subunit gene and several known transcript variants of skeletal, cardiac and brain beta genes. The mdg mutation is retained in the 129DA3 cell line and occurs exclusively at nucleotide position 4010 in the skeletal alpha 1 transcript in which a cytosine residue is deleted. In early stages of differentiation and fusion, Ba2+ currents were detected in dysgenic myotubes the same as the cardiac L-type Ca2+ channel. These data provide specific structural evidence [Chaudhari, N. (1992) J. Biol. Chem. 267, 25636-25639] for the major genetic defect in mouse muscular dysgenesis and show a change in the expression levels of alpha 1S and alpha 1C. The upregulation of the expression of alpha 1C results in functional Ca2+ channel activity, however, presumably not sufficient for excitation-contraction coupling.

Reversion of malignant phenotype by 5-iodo-6-amino-1,2-benzopyrone a non-covalently binding ligand of poly(ADP-ribose) polymerase.

A non-covalently binding inhibitory ligand of poly(ADP-ribose) polymerase, 5-iodo-6-amino-1,2-benzopyrone, when incubated at 5-600 microM external concentration with an E-ras-transformed tumorigenic cell line or with human prostatic carcinoma cells for 40 to 60 days converts both cancer cells to a non-tumorigenic phenotype that is characterized by drastic changes in cell morphology, absence of tumorigenicity in nude mice, and a high rate of aerobic glycolysis.

Contribution of kinases and the CD45 phosphatase to the generation of tyrosine phosphorylation patterns in the T-cell receptor complex zeta chain.

The zeta subunit of the T-cell receptor complex plays a crucial role in coupling the antigen binding alphabeta and gammadelta heterodimers to the downstream activation pathways. Three tandem amino acid sequence motifs containing pairs of exactly spaced Tyr-X-X-Leu/Ile sequences, designated as Immunoreceptor Tyrosine-based Activation Motifs (ITAMs), control this function. The phosphorylated forms of ITAMs serve as docking sites for several src homology 2 (SH2) domain containing signaling proteins. The composition of the assembled signaling complex and the outcome of cell activation depends on the tyrosine phosphorylation pattern of the zeta polypeptide. The mechanism that conducts the generation of various phosphorylated forms has not yet been well established. In this study we have analyzed the ability of src family tyrosine kinases and the CD45 tyrosine phosphatase in determining the phosphorylation state of the different ITAMs and the individual tyrosine residues of the TCR zeta chain. The intracellular part of the zeta chain was phosphorylated by src family tyrosine kinases, p56lck and p59fyn in vitro. Synthetic oligopeptides representing full-length or half-sized ITAMs with a single tyrosine residue were also phosphorylated by both p56lck and p59fyn. In contrast, an additional membrane proximal tyrosine residue in the human zeta chain, located outside of the ITAMs, was not phosphorylated. We also examined the activity of the CD45 phosphatase, using a panel of ITAM derivatives, in which one or both tyrosines were phosphorylated. The efficiency of ITAM dephosphorylation by CD45 was dependent on the primary sequence of the oligopeptides and the position of the phosphotyrosine residues. The in vitro data suggest that the CD45 phosphatase rather than the tyrosine kinase(s) may control the generation of specific phosphorylation patterns of the zeta chain during cell activation.

The role of thiotepa in allogeneic stem cell transplantation in patients with leukemia.

Graft versus host disease (GVHD) and recurrence of basic disease are major obstacles to a successful allogeneic bone marrow transplantation (BMT) outcome. One of the possibilities of maintaining the therapeutic potential of marrow allografting in the absence of GVHD is to intensify the conditioning regimen administered pre-T-cell depleted BMT in order to compensate for the loss of GVH related graft versus leukemia (GVL) effect. In order to do so we used a preparative regimen consisting of three alkylating agents-Busulfan (BU), Thiotepa (TTP) and Cyclophosphamide (CY)-for T-cell depleted allogeneic stem cell transplantation (SCT) instead of the standard BU-CY protocol. The effect of this intensified regimen was investigated in 30 consecutive leukemia patients who underwent T-cell depleted SCT from HLA identical siblings. Sixteen of the patients were males and 14 females, of median age 24 (5-43) years. Fourteen patients had acute myelogenous leukemia (AML), ten acute lymphoblastic leukemia (ALL), four chronic myelogenous leukemia (CML) and two myelodysplastic syndrome. The conditioning regimen consisted of BU 4 mg/kg x 4 days (-8 to -5), TTP 5 mg/kg x 2 days (-4 and -3), and CY 60 mg/kg x 2 days (-2 and -1). Engraftment was normal, with WBC >1.0x10(9)/l at day +18 (10-32), ANC >0.5x10(9)/l at day +21 (9-33) and platelets >25x10(9)/l at day +30 (14-69). Regimen related toxicity (RRT) was moderate and transplant related complications comparable to other conventional conditioning protocols. Overall survival and disease free survival (DFS) at 60 months follow up was 50%. Only three patients (10%), with ALL, relapsed and subsequently died. From the current data it would appear that TTP does not significantly improve BMT outcome in patients with leukemia, when compared to the standard BU-CY conditioning. However, our results with the BU-TTP-CY combination followed by T-cell depleted allogeneic SCT could provide the basis for a prospective randomized study comparing this protocol with the standard BU-CY regimen.